Both plants and animals have adapted networks of vessels or channels for convective transport of nutrients and waste to overcome the limits of diffusion. Hence, the successful creation of large (order 1 cm or larger) implantable tissues will necessarily require mechanisms of transport other than simple diffusion. The past two decades have brought enormous understanding of basic biological mechanisms, and, as a result the promise of new therapies aimed at replacing or repairing damaged tissues. This field has been dubbed "regenerative medicine" or "tissue engineering", and, although bursting with potential, progress has been slowed due, in large part, to a lack of solutions for achieving adequate transport of nutrients and waste in thicker tissues. While several approaches have been proposed, it is our premise that a biology-directed strategy will prove the most successful. We propose to prevascularize in vitro a thick tissue with an interconnected network of mature fully-formed human microvessels (with supporting pericytes) prior to implantation. Upon implantation to the host, the continuous network of microvessels is primed for rapid anastomosis and perfusion of the tissue thereby maintaining viability. Being composed of true human microvessels, the dynamic vascular network can then remodel (prune or extend, become arterioles or venules) in response to the metabolic needs of the tissues. Our published and preliminary data demonstrate the feasibility of this approach including perfusion of the prevascularized tissue with host blood within ~ 24 hours of implantation. However, we have yet to demonstrate the efficacy in a truly thick (~ 1 cm) tissue that contains hypoxia-sensitive cells. Hence, our proposal has two specific aims: 1) using endothelial cells derived from endothelial precursor cells (EPC-EC) from either cord or adult peripheral blood, and an appropriate stromal cell (e.g., fibroblast), create a thick ( ~ 1 cm) tissue that is prevascularized with a continuous interconnected network of well-formed microvessels supported by pericytes, containing a hypoxia-sensitive indicator cell (e.g., cardiac myocyte) distributed throughout, and suitable for implantation;2) establish rapid (<24 hours) anastomosis with the host (immune- compromised mouse) circulation and perfusion to maintain tissue viability. When successful, the results of this proposed R21 will lay the groundwork for numerous larger scale projects including the incorporation of tissue specific functionality. PUBLIC HEALTH RELEVANCE: This project will design a thick (1 cm) implantable prevascularized tissue which contains 1) a mature interconnected network of human microvessels, 2) supporting pericytes, and 3) a hypoxia-sensitive cell (e.g., cardiac myocyte). We seek to demonstrate rapid (<24 hours) anastomosis with the host circulation and superior cell survivability, which will have broad applications in the field of regenerative medicine.